A zinc-dependent nuclear endonuclease is responsible for DNA laddering during salt-induced programmed cell death in root tip cells of rice

DNA laddering is one of the biochemical processes characteristic of programmed cell death (PCD) both in animals and plants. However, the mechanism of DNA laddering varies in different species, even in different tissues of one organism. In the present study, we used root tip cells of rice, which have been induced by NaCl stress to undergo PCD, to analyze the endonuclease activities of cytoplasmic and nuclear extracts. Two endonucleases, a cytoplasmic of 20kDa (OsCyt20) and a nuclear of 37kDa (OsNuc37), were identified as PCD related. Our results indicated that OsCyt20 is a Ca(2+)/Mg(2+)-dependent nuclease, which is most active at neutral pH, and that OsNuc37 is Zn(2+)-dependent, with a pH optimum of 4.5-6. Both nucleases were induced at the early stage of PCD (2h salt treatment) and exhibited the highest activity approximately 4h after exposure to NaCl, paralleling with the occurrence of DNA laddering. In vitro assays of endonuclease activities further revealed that OsNuc37, a glycoprotein localized in the nucleus, is the executor for DNA laddering. The different effects of both endonucleases on DNA degradation during salt-induced PCD are discussed.     

Characterization of nuclease activities and DNA fragmentation induced upon hypersensitive response cell death and mechanical stress

Programmed cell death (PCD) is activated during the response of multicellular organisms to some invading pathogens. One of the key aspects of this process is the degradation of nuclear DNA which is thought to facilitate the recycling of DNA from dead cells. The PCD of tobacco plants (genotype NN) infected with tobacco mosaic virus (TMV) is accompanied by the induction of nuclease activities and the cleavage of nuclear DNA to fragments of about 50 kb. We examined the correlation between the increase in nuclease activities and the fragmentation of nuclear DNA during TMV- and bacteria-induced PCD in tobacco. We found that the increase in nuclease activities did not always correlate with fragmentation of nuclear DNA. Thus, in addition to pathogens that induce PCD, mechanical injury and infiltration of leaves with 1 M sucrose or bacteria that did not induce PCD also resulted in an increase in nuclease activities. Analysis of nuclease activities in total leaf extracts, nuclear extracts, and intercellular fluid (i.e., apoplast) revealed that at least four different nuclease activities are induced during PCD in tobacco; of these at least three appear to be secreted into the intercellular fluid. Although the latter were also induced in response to treatments that did not result in DNA fragmentation, they may function in the recycling of plant DNA during late stages of PCD when the integrity of the plasma membrane is compromised. This suggestion is supported by the finding that DNA degradation occurred late during TMV-induced PCD in tobacco. In addition, the finding of induced nuclease activities in the intercellular fluid raises the possibility that they may serve a protective function by degrading the DNA of invading pathogens.     

The nucellus degenerates by a process of programmed cell death during the early stages of wheat grain development

The nucellus, which is the maternal tissue of the wheat grain, degenerates during the early stages of development. We have investigated whether or not this degenerative process may be considered as programmed cell death (PCD). The analysis of DNA of tissues dissected from developing wheat (Triticum aestivum L. cv Chinese Spring) grains at 5-20 days post anthesis (dpa) showed the presence of DNA laddering, which is indicative of internucleosomal fragmentation of nuclear DNA, in maternal tissues but not in the endosperm. The TUNEL assay showed in-situ internucleosomal fragmentation of DNA in nuclei of parenchymal and epidermal cells of the nucellus, as well as in the pericarp, during the early stages of grain development (5 dpa). Furthermore, internucleosomal fragmentation of nuclear DNA was observed in nucellar projection cells in the middle stages of grain development (13-18 dpa), thus showing a process of PCD in these maternal tissues. Electron-transmission microscopy analysis allowed the morphology of PCD to be characterized in this plant tissue. Initially, fragmentation of the cytoplasm was observed, the nuclear envelope appeared dilated and to be forming vacuoles, and the content of heterochromatin increased. A progressive degradation of the cytosolic contents and organelles was observed, and the plasma membrane was disrupted. However, the Golgi apparatus remained intact and apparently functional even in the final stages of cell death.     

DNA degradation and nuclear degeneration during programmed cell death in petals of Antirrhinum, Argyranthemum, and Petunia

Programmed cell death (PCD) was studied in the petals of Antirrhinum majus, Argyranthemum frutescens, and Petunia hybrida, using DNA degradation and changes in nuclear morphology as parameters. The petals exhibit loss of turgor (wilting) as a visible symptom of PCD. DNA degradation, as shown on agarose gels, occurred in all species studied, prior to visible wilting. The number of DNA masses in all the petals of a flower, determined by flow cytometry, markedly increased in Argyranthemum and Petunia, but decreased in Antirrhinum. Many small DNA masses were observed in Argyranthemum and Petunia. The surface of each small DNA mass stained with the lipophilic fluorochrome 3,3'-dihexyloxacarbocyanine iodide (DiOC6), indicating that these masses were surrounded by a membrane. In Antirrhinum, in contrast, the chromatin fragmented into several small spherical clumps that remained inside a large membranous structure. Nuclear fragmentation, therefore, did not occur in Antirrhinum, whereas nuclear fragmentation possibly was a cause of the small DNA masses in Argyranthemum and Petunia. It is concluded that at least two contrasting nuclear morphologies exist during PCD. In the first, the chromatin fragments inside the nucleus, not accompanied--or followed--by nuclear fragmentation. In the second, a large number of DNA masses were observed each enveloped by a membrane. The second type was probably due, at least partially, to nuclear fragmentation.     

Apoptotic DNA fragmentation is detected by a semi-quantitative ligation-mediated PCR of blunt DNA ends

Apoptosis is a form of programmed cell death (PCD) characterized by morphological changes and stereotypical DNA degradation described as a nucleosomal ;ladder'. However, nucleosomal ladders have only been clearly demonstrated in vertebrate tissues when large numbers of cells die in synchrony. Their absence may be explained by asynchronous death under physiological conditions, or by distinct molecular mechanisms. In this study, nucleosomal ladders were revealed by a ligation-mediated polymerase chain reaction (LMPCR), that amplifies DNA fragments with blunt, 5' phosphorylated ends. Numerous tissues from different organisms were examined which demonstrated that nucleosomal ladders (a) accompany physiological cell death in mammalian tissues where previously DNA fragmentation has not been detected; (b) are produced during invertebrate cell death; (c) are invariably generated via the production of blunt, 5' phosphorylated double strand breaks. These results suggest that PCD in multicellular organisms consistently involves apoptotic mechanisms and that the endonuclease activity is evolutionarily conserved.     

ABCA1-dependent but apoA-I-independent cholesterol efflux mediated by fatty acid-bile acid conjugates (FABACs)

PCD (programmed cell death) in plants presents important morphological and biochemical differences compared with apoptosis in animal cells. This raises the question of whether PCD arose independently or from a common ancestor in plants and animals. In the present study we describe a cell-free system, using wheat grain nucellar cells undergoing PCD, to analyse nucleus dismantling, the final stage of PCD. We have identified a Ca2+/Mg2+ nuclease and a serine protease localized to the nucleus of dying nucellar cells. Nuclear extracts from nucellar cells undergoing PCD triggered DNA fragmentation and other apoptotic morphology in nuclei from different plant tissues. Inhibition of the serine protease did not affect DNA laddering. Furthermore, we show that the nuclear extracts from plant cells triggered DNA fragmentation and apoptotic morphology in nuclei from human cells. The inhibition of the nucleolytic activity with Zn2+ or EDTA blocked the morphological changes of the nucleus. Moreover, nuclear extracts from apoptotic human cells triggered DNA fragmentation and apoptotic morphology in nuclei from plant cells. These results show that degradation of the nucleus is morphologically and biochemically similar in plant and animal cells. The implication of this finding on the origin of PCD in plants and animals is discussed.     

Programmed cell death in procyclic Trypanosoma brucei rhodesiense is associated with differential expression of mRNAs

Procyclic Trypanosoma brucei rhodesiense have a cell death mechanism which can be activated by an external signal, the lectin ConA, in vitro. ConA has been shown to cause profound changes in cellular morphology and induce fragmentation of nuclear DNA in T.b. rhodesiense which are characteristic of apoptosis, a form of programmed cell death (PCD) in other eukaryotic cells. RNA analysis of trypanosomes induced to undergo PCD revealed that RNA remains intact up to 48 h into the process, a time when nuclear DNA fragmentation has already started. Using the randomly amplified differentially expressed sequences polymerase chain reaction method, ConA-induced cell death in T.b. rhodesiense is shown to be associated with differential expression of mRNAs, including up regulation of mRNAs late in the death process. The results demonstrate that trypanosomes actively participate in their own destruction through a PCD process and confirm that cell death in trypanosomes is associated with de novo gene expression.     

The PET1-CMS mitochondrial mutation in sunflower is associated with premature programmed cell death and cytochrome c release

In mammals, mitochondria have been shown to play a key intermediary role in apoptosis, a morphologically distinct form of programmed cell death (PCD), for example, through the release of cytochrome c, which activates a proteolytic enzyme cascade, resulting in specific nuclear DNA degradation and cell death. In plants, PCD is a feature of normal development, including the penultimate stage of anther development, leading to dehiscence and pollen release. However, there is little evidence that plant mitochondria are involved in PCD. In a wide range of plant species, anther and/or pollen development is disrupted in a class of mutants termed CMS (for cytoplasmic male sterility), which is associated with mutations in the mitochondrial genome. On the basis of the manifestation of a number of morphological and biochemical markers of apoptosis, we have shown that the PET1-CMS cytoplasm in sunflower causes premature PCD of the tapetal cells, which then extends to other anther tissues. These features included cell condensation, oligonucleosomal cleavage of nuclear DNA, separation of chromatin into delineated masses, and initial persistence of mitochondria. In addition, immunocytochemical analysis revealed that cytochrome c was released partially from the mitochondria into the cytosol of tapetal cells before the gross morphological changes associated with PCD. The decrease in cytochrome c content in mitochondria isolated from male sterile florets preceded a decrease in the integrity of the outer mitochondrial membrane and respiratory control ratio. Our data suggest that plant mitochondria, like mammalian mitochondria, play a key role in the induction of PCD. The tissue-specific nature of the CMS phenotype is discussed with regard to cellular respiratory demand and PCD during normal anther development.     

Aspects of programmed cell death during leaf senescence of mono- and dicotyledonous plants

Summary Leaf senescence is a highly regulated stage in the plant life cycle, leading to cell death, recently examined as a type of the programmed cell death (PCD). One of the basic features of PCD is the condensation of nuclear chromatin which is caused by endonucleolytic degradation of nuclear DNA (nDNA). In our investigations, we applied the technique of the single-cell electrophoresis system (“comet assay”) in order to determine the type of nDNA fragmentation during leaf senescence. The comet assay, a sensitive method revealing nonrandom internucleosomal damage that is specific for PCD, is especially useful for the detection of nDNA degradation in isolated viable cells. Simultaneously, we analyzed the mesophyll cell ultrastructure and the photosynthetic-pigment concentration in the leaves of two species,Ornithogalum virens andNicotiana tabacum, representing mono- and dicotyledonous plants which differ in the pattern of leaf differentiation. These investigations demonstrated that, in both species, the comet assay revealed nDNA degradation in yellow-leaf protoplasts containing chloroplasts that showed already changed ultrastructure (swelled or completely degraded thylakoids) and cell nuclei with a significant condensation of chromatin. There was no nDNA degradation in green-leaf protoplasts containing differentiated chloroplasts with numerous grana stacks and nuclei with dispersed chromatin. The analysis of intermediate developmental stage showed that the degradation of nDNA precedes condensation of nuclear chromatin. Thus the comet assay is a very useful and sensitive method for early detection of PCD. Moreover, results of our studies indicate that leaf senescence involves PCD.     

ZEN1 is a key enzyme in the degradation of nuclear DNA during programmed cell death of tracheary elements

Tracheary elements (TEs) have a unique cell death program in which the rapid collapse of the vacuole triggers the beginning of nuclear degradation. Although various nucleases are known to function in nuclear DNA degradation in animal apoptosis, it is unclear what hydrolase is involved in nuclear degradation in plants. In this study, we demonstrated that an S1-type nuclease, Zinnia endonuclease 1 (ZEN1), functions directly in nuclear DNA degradation during programmed cell death (PCD) of TEs. In-gel DNase assay demonstrated the presence of a 24-kD Ca(2+)/Mg(2+)-dependent nuclease and a 40-kD Zn(2+)-dependent nuclease as well as ZEN1 in 60-h-cultured cells that included differentiating TEs. Such cell extracts possessed the ability to degrade the nuclear DNA isolated from Zinnia elegans cells in the presence of Zn(2+), and its activity was suppressed by an anti-ZEN1 antibody, indicating that ZEN1 is a central DNase responsible for nuclear DNA degradation. The introduction of the antisense ZEN1 gene into Zinnia cells cultured for 40 h specifically suppressed the degradation of nuclear DNA in TEs undergoing PCD but did not affect vacuole collapse. Based on these results, a common mechanism between animal and plant PCD is discussed.     

Identification of programmed cell death in situ in individual plant cells in vivo using a chromosome preparation technique

A simple procedure, which combines a chromosome preparation technique with an in situ labelling technique modified from fluorescence in situ hybridization (FISH), has been developed for in situ detection of plant programmed cell death (PCD) at the single-cell level. After exposure of chromosomes and nuclei on slides by enzymolysis, Klenow or TdT was used to incorporate Bio-dUTP or fluorescein-dUTP at sites of DNA breaks. After Klenow-mediated labelling, the signals were amplified by a cascade of antigen-antibody reaction according to the detection system of FISH. This method enables in situ detection of plant PCD in vivo morphologically and biochemically at the chromosome, nuclear and DNA levels without cell culture and histological sectioning. This technique permits labelling of DNA breaks with high sensitivity due to increased chromosome and nucleus exposure to the labelling solutions, as well as due to the immunological amplification of the signals. Moreover, the changes in the cells were easier to be observed because the spatial obstacle of the cell wall and its autofluorescence were eliminated. It is potentially useful for in situ detection of PCD in plant root meristematic cells triggered by various environmental abiotic factors. It is proposed that the root tip is a versatile in vivo system for studying PCD induced by environmental abiotic factors.     

Proteomics analysis of rice lesion mimic mutant (spl1) reveals tightly localized probenazole-induced protein (PBZ1) in cells undergoing programmed cell death

Numerous reports have predicted/hypothesized a role for probenazole-induced protein (PBZ1) as a molecular marker in rice self-defense mechanism. However, the precise function of PBZ1 remains unknown. In the present study, we examined PBZ1 as a putative cell death marker in rice. For this, we focused our attention on a rice lesion mimic mutant (LMM), spotted leaf 1 ( spl1), which has been used to study the programmed cell death (PCD) phenomenon during lesion development in leaf. Using two-dimensional gel electrophoresis (2-DGE), 18 colloidal Coomassie brilliant blue stained protein spots were found to be differentially expressed in the leaves of spl1 mutant. After analysis of these spots by MALDI-TOF-MS, we identified the PBZ1 protein to be highly inducible in spl1. On the basis of these results, we proceeded to verify whether PBZ1 is highly expressed in the tissues undergoing PCD in rice. To do so, we performed immunoblot analysis and immunolocalization and used transgenic lines carrying the PBZ1 promoter fused with GFP. Results demonstrated that the expression levels and localizations of PBZ1 dramatically coincided with tissues undergoing PCD, namely, during leaf senescence, root aerenchyma formation, coleoptiles senescence, root cap, and seed aleurone layer. Furthermore, localization of the PBZ1 protein was also tightly correlated with TUNEL signal in the seed aleurone layer. As DNA fragmentation is a hallmark of PCD, this result clearly indicates a role for PBZ1 in rice tissues undergoing PCD. In conclusion, our results provide strong support for the hypothesis that PBZ1 is a molecular marker in rice defense response, and can serve as a novel potential marker for cell death/PCD in rice.     

Endonucleases and their involvement in plant apoptosis

This review considers modern data about the set, nature, specificity of action, and other properties of plant endonucleases involved in various forms of programmed cell death (PCD) in various plant tissues (organs). Apoptosis is an obligatory component of plant development; plant development is impossible without apoptosis. In dependence on the conditions of plant growth, this process can be induced by various biotic and abiotic factors, including stressors. Endonucleases accomplishing apoptotic degradation of nuclear material in the plant cell play one of the main roles in PCD. Plant endonucleases belong to at least two classes: (1) Ca²⁺- and Mg²⁺-dependent and (2) Zn²⁺-dependent nucleases. The set and activities of endonucleases change with plant age and during apoptosis in a tissue-specific manner. Apoptosis is accompanied by the induction of specific endonucleases hydrolyzing DNA in chromatin with the formation firstly of large domains and then internucleosomal DNA fragments; the products produced are of about 140 nucleotides in length with their subsequent degradation to low-molecular-weight oligonucleotides and mononucleotides. About 30 enzymes are involved in apoptotic DNA degradation. Histone H1 modulates endonuclease activity; separate (sub)fractions of this nuclear protein can stimulate or inhibit corresponding plant endonucleases. In the nucleus and cytoplasm of the plant cells, Ca²⁺/Mg²⁺-dependent endonucleases recognizing substrate DNA methylation status were revealed and described for the first time; their action resembles that of bacterial restrictases, which activity is modulated by the donor of methyl groups, S-adenosylmethionine. This indicates that higher eukaryotes (higher plants) might possess the system of restriction-modification to some degree analogous to that of prokaryotes.     

Leaf‐shape remodeling: programmed cell death in fistular leaves of Allium fistulosum

Some species of Allium in Liliaceae have fistular leaves. The fistular lamina of Allium fistulosum undergoes a process from solid to hollow during development. The aims were to reveal the process of fistular leaf formation involved in programmed cell death (PCD) and to compare the cytological events in the execution of cell death to those in the unusual leaf perforations or plant aerenchyma formation. In this study, light and transmission electron microscopy were used to characterize the development of fistular leaves and cytological events. Terminal deoxynucleotidyl transferase‐mediated dUTP nick end labeling (TUNEL) assays and gel electrophoresis were used to determine nuclear DNA cleavage during the PCD. The cavity arises in the leaf blade by degradation of specialized cells, the designated pre‐cavity cells, in the center of the leaves. Nuclei of cells within the pre‐cavity site become TUNEL‐positive, indicating that DNA cleavage is an early event. Gel electrophoresis revealed that DNA internucleosomal cleavage occurred resulting in a characteristic DNA ladder. Ultrastructural analysis of cells at the different stages showed disrupted vacuoles, misshapen nuclei with condensed chromatin, degraded cytoplasm and organelles and emergence of secondary vacuoles. The cell walls degraded last, and residue of degraded cell walls aggregated together. These results revealed that PCD plays a critical role in the development of A. fistulosum fistular leaves. The continuous cavity in A. fistulosum leaves resemble the aerenchyma in the pith of some gramineous plants to improve gas exchange.     

Programmed cell death progressively models the development of anther sporophytic tissues from the tapetum and is triggered in pollen grains during maturation

To characterize the spatial and temporal occurrence of programmed cell death (PCD) in Lilium anther tissues, we used both microscopical and molecular markers of apoptosis for developmental stages from meiosis to pollen release. The first hallmarks of PCD include cell condensation and shrinkage of the cytoplasm, separation of chromatin into delineated masses, and DNA fragmentation in the tapetum as early as the premeiosis stage. PCD then extended to other anther sporophytic tissues, leading to anther dehiscence. Although the PCD clearly affected the endothecium and the epidermis, these two cell layers remained alive until anther dehiscence. In pollen, no sign of PCD was found until pollen mitosis I, after what apoptotic features developed progressively in the vegetative cell. In addition, DNA ladders were detected in all sporophytic tissues and cell types throughout pollen development, whereas in the male gametophyte DNA ladders were only detected during pollen maturation. Our data suggest that PCD is a progressive and active process affecting all the anther tissues, first being triggered in the tapetum.     

Programmed cell death induced by (β-d-galactosyl)3 Yariv reagent in Nicotiana tabacum BY-2 suspension-cultured cells

Programmed cell death (PCD) is involved in plant development and pathogen defence and can be triggered in vitro by several biotic and abiotic stimuli. In this report ( β - d -galactosyl)₃ Yariv reagent, a chemical that specifically binds to arabinogalactan-proteins (AGPs), completely inhibited cell growth and induced PCD in tobacco BY-2 suspension cultured cells. Analysis of DNA from these cells, by agarose gel electrophoresis, revealed a DNA ladder consisting of multimers of 140–170 bp, similar to apoptotic animal DNA internucleosomal fragmentation. Complementary morphological studies revealed additional PCD characteristics in the Yariv-treated BY-2 cells, including cell shrinkage and cytoplasmic condensation. These studies demonstrate the usefulness of BY-2 cells as a model plant PCD system and confirm a link between AGPs and PCD.     

The scutellum of germinated wheat grains undergoes programmed cell death: identification of an acidic nuclease involved in nucleus dismantling

Programmed cell death (PCD) is a crucial phenomenon in the life cycle of cereal grains. In germinating grains, the scutellum allows the transport of nutrients from the starchy endosperm to the growing embryo, and therefore it may be the last grain tissue to undergo PCD. Thus, the aim of this work was to analyse whether the scutellum of wheat grains undergoes PCD and to perform a morphological and biochemical analysis of this process. Scutellum cells of grains following germination showed a progressive increase of DNA fragmentation, and the TUNEL assay showed that PCD extended in an apical-to-basal gradient along the scutellum affecting epidermal and parenchymal cells. Electron-transmission microscopy revealed high cytoplasm vacuolation, altered mitochondria, and the presence of double-membrane structures, which might constitute symptoms of vacuolar cell death, whereas the nucleus appeared lobed and had an increased heterochromatin content as the most distinctive features. An acid- and Zn(2+)-dependent nucleolytic activity was identified in nuclear extracts of scutellum cells undergoing PCD. This nuclease was not detected in grains imbibed in the presence of abscisic acid, which inhibited germination. This nucleolytic activity promoted DNA fragmentation in vitro on nuclei isolated from healthy cells, thus suggesting a main role in nucleus dismantling during PCD.     

Cytofluorometric and cytochemical comparisons of normal and abnormal human cells from the female genital tract.

Acridine orange staining of exfoliated cells from epithelial tissues facilitates discrimination between normal and abnormal cells: abnormal cells develop highly elevated nuclear fluorescence. Comparisons of acridine orange (AO) staining with propidium iodide (PI) or Feulgen staining have shown that: (a) PI staining also provides highly elevated nuclear fluorescence from abnormal cells; (b) the distributions of nuclear fluorescence following AO or PI staining were usually not significantly different as judged by the Kolmogorov-Smirnov test; (c) fluorescence emission spectra from AO and PI stained cells are consistent with the hypothesis that both fluorochromes bind to DNA within cell nuclei; (d) DNAse treatment of AO stained normal cells eliminates the nuclear fluorescence peak from slit-scan contours; RNAse treatment has no effect on nuclear fluorescence; (e) the distribution of abnormal cell nuclear fluorescence after AO staining is usually, but not always, significantly different from the distribution of abnormal cell nuclear absorbance after Feulgen staining, with relative nuclear fluorescence being greater than relative nuclear absorbance. The hypothesis currently most consistent with these results is that elevated Feulgen DNA content can account for only part of the discrimination provided by AO staining, and that the chromatin within abnormal cells is altered so as to increase accessibility of DNA to intercalating dyes.     

DNA degradation during programmed cell death in Phaseolus coccineus suspensor.

The suspensor of Phaseolus coccineus L. degenerates at the cotyledonary stage of embryogenesis when it is no longer necessary for continued embryonic development; this degeneration is considered to be a typical example of the so-called developmental programmed cell death (PCD) in plants. The presence of specific hallmarks of PCD as it occurs during the degeneration of P. coccineus suspensor was investigated in the current study. By using the TUNEL assay and electrophoretic analysis, we found evidence of nuclear DNA degradation, a known feature of PCD, in the endosperm and degenerating suspensors. Degeneration of the suspensor begins after degeneration in the endosperm and it starts in the neck region, spreading basipetally towards the knob. We conclude from this study that suspensor degeneration in P. coccineus occurs by means of PCD and displays typical hallmarks of PCD, such as DNA fragmentation. PCD in the suspensor is a highly asynchronous process, originating first in the neck cells and subsequently spreading to the basal cells.     

Use of LysoTracker to Detect Programmed Cell Death in Embryos and Differentiating Embryonic Stem Cells

Programmed cell death (PCD) occurs in adults to maintain normal tissue homeostasis and during embryological development to shape tissues and organs^1,2,6,7. During development, toxic chemicals or genetic alterations can cause an increase in PCD or change PCD patterns resulting in developmental abnormalities and birth defects^3-5. To understand the etiology of these defects, the study of embryos can be complemented with in vitro assays that use differentiating embryonic stem (ES) cells.Apoptosis is a well-studied form of PCD that involves both intrinsic and extrinsic signaling to activate the caspase enzyme cascade. Characteristic cell changes include membrane blebbing, nuclear shrinking, and DNA fragmentation. Other forms of PCD do not involve caspase activation and may be the end-result of prolonged autophagy. Regardless of the PCD pathway, dying cells need to be removed. In adults, the immune cells perform this function, while in embryos, where the immune system has not yet developed, removal occurs by an alternative mechanism. This mechanism involves neighboring cells (called "non-professional phagocytes") taking on a phagocytic role-they recognize the ''eat me'' signal on the surface of the dying cell and engulf it^8-10. After engulfment, the debris is brought to the lysosome for degradation. Thus regardless of PCD mechanism, an increase in lysosomal activity can be correlated with increased cell death.To study PCD, a simple assay to visualize lysosomes in thick tissues and multilayer differentiating cultures can be useful. LysoTracker dye is a highly soluble small molecule that is retained in acidic subcellular compartments such as the lysosome^11-13. The dye is taken up by diffusion and through the circulation. Since penetration is not a hindrance, visualization of PCD in thick tissues and multi-layer cultures is possible^12,13. In contrast, TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) analysis¹⁴, is limited to small samples, histological sections, and monolayer cultures because the procedure requires the entry/permeability of a terminal transferase.In contrast to Aniline blue, which diffuses and is dissolved by solvents, LysoTracker Red DND-99 is fixable, bright, and stable. Staining can be visualized with standard fluorescent or confocal microscopy in whole-mount or section using aqueous or solvent-based mounting media^12,13. Here we describe protocols using this dye to look at PCD in normal and sonichedgehog null mouse embryos. In addition, we demonstrate analysis of PCD in differentiating ES cell cultures and present a simple quantification method. In summary, LysoTracker staining can be a great complement to other methods of detecting PCD.